Escherichia coli specifically incorporate selenium into tRNA. Forty percent of the selenium-containing tRNA had lysine acceptor activity and an additional 10% had glutamate acceptor activity. About 5% of the total tRNA-lys and 1% of the total tRNA-glu was selenium-modified. Analysis by high performance liquid chromatography (HPLC) indicated the presence of only one selenium-containing nucleoside. The UV absorbance spectrum and elution position during HPLC of this selenonucleoside identify it as 5-methylaminomethyl-2-selenouridine, a previously uncharacterized compound.